Extracellular matrix regulation of differentiation via modulation of ILK: application to 3D bioprinting of cardiac tissue

通过调节 ILK 进行细胞外基质分化调节：在心脏组织 3D 生物打印中的应用

基本信息

批准号：
9301966
负责人：
Michael McAlpine
金额：
$ 37.44万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9301966
关键词：
3D Print Accounting Activities of Daily Living Acute Address Adult Arteries Behavior Biochemistry Biological Biological Models Bioreactors Blood Vessels Cardiac Cardiac Myocytes Cardiac development Cardiovascular Diseases Cardiovascular Physiology Cause of Death Cell Differentiation process Cells Cessation of life Chemicals Chronic Collagen Type I Core Facility Couples Cyclin-Dependent Kinase Inhibitor 3 Dependence Deposition Developed Countries Developing Countries Dimensions Disease model Elements Endothelial Cells Engineering Event Extracellular Matrix Extracellular Matrix Proteins Fibronectins Focal Adhesion Kinase 1 Focal Adhesions Formulation Gene Expression Generations Genes Genetic Transcription Goals Heart Heart Transplantation Human In Vitro Individual Injury Integrins Laboratories Ligation Link Literature Mass Spectrum Analysis Mediating Mesoderm Molecular Motivation Mus Myocardial Myocardial tissue Myocarditis Myocardium Natural regeneration Nutrient Outcome Oxygen Pathway interactions Perfusion Pluripotent Stem Cells Preclinical Drug Evaluation Printing Proteins RNA Interference Regulation Research Signal Pathway Signal Transduction Spatial Design Stem cells Technology Testing Therapeutic Thick Time Tissue Engineering Tissues Toxicity Tests Transcriptional Activation United States arteriole base beta catenin bioprinting cell behavior cell type design glycogen synthase kinase 3 beta inhibitor in vivo induced pluripotent stem cell innovation insight integrin-linked kinase kinase inhibitor matrigel mechanical force meetings muscle form optical imaging organ regeneration programs stem cell differentiation tissue repair tool transcription factor trend

3D Print Accounting Activities of Daily Living Acute Address Adult Arteries Behavior Biochemistry Biological Biological Models Bioreactors Blood Vessels Cardiac Cardiac Myocytes Cardiac development Cardiovascular Diseases Cardiovascular Physiology Cause of Death Cell Differentiation process Cells Cessation of life Chemicals Chronic Collagen Type I Core Facility Couples Cyclin-Dependent Kinase Inhibitor 3 Dependence Deposition Developed Countries Developing Countries Dimensions Disease model Elements Endothelial Cells Engineering Event Extracellular Matrix Extracellular Matrix Proteins Fibronectins Focal Adhesion Kinase 1 Focal Adhesions Formulation Gene Expression Generations Genes Genetic Transcription Goals Heart Heart Transplantation Human In Vitro Individual Injury Integrins Laboratories Ligation Link Literature Mass Spectrum Analysis Mediating Mesoderm Molecular Motivation Mus Myocardial Myocardial tissue Myocarditis Myocardium Natural regeneration Nutrient Outcome Oxygen Pathway interactions Perfusion Pluripotent Stem Cells Preclinical Drug Evaluation Printing Proteins RNA Interference Regulation Research Signal Pathway Signal Transduction Spatial Design Stem cells Technology Testing Therapeutic Thick Time Tissue Engineering Tissues Toxicity Tests Transcriptional Activation United States arteriole base beta catenin bioprinting cell behavior cell type design glycogen synthase kinase 3 beta inhibitor in vivo induced pluripotent stem cell innovation insight integrin-linked kinase kinase inhibitor matrigel mechanical force meetings muscle form optical imaging organ regeneration programs stem cell differentiation tissue repair tool transcription factor trend

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项目摘要

PROJECT SUMMARY The primary objective of this proposal is to couple a) mechanistic insight relating differentiation outcomes to ECM engagement via intracellular signaling events triggered at the focal adhesion (FA), with b) 3D printing of ECM and ECM-associated proteins as a means to direct cell distribution with maturation and thereby enable fabrication of thick, functional cardiac tissue. The proposal is significant as it has the potential to generate replacement tissues and even heart grafts for individuals suffering from acute and chronic injury to the heart. It is the first of its kind to address the conundrum of the apparent uniformity of the focal adhesion relative to the myriad of different ECM/integrin combinations and the corresponding variety of cell behaviors that emerge from ECM engagement. It does so by proposing that elements of the FA, namely integrin linked kinase (ILK) and associated phosphatase, act as sensitive rheostats that can be co-opted to yield desired behavior. Here, the desired behavior is cardiac differentiation, and the innovation is the utilization of optimized ECM formulations as bioinks to create 3D cardiac tissue mimics (3DCTM) capable of directing cell distribution of multiple cell types with differentiation. This concept is feasible as the Ogle laboratory has long-studied the biochemistry of the ECM and stem cell behaviors associated with ECM engagement, and the McAlpine laboratory has focused on 3D printing of functional materials for a range of applications, from biological to electronic and the merger of these materials. These groups will also interface with expertise of the Kamp lab with respect to their recent generation of induced cardiac progenitor cells (iCPCs) to populate the 3DCTM, the Provenzano lab to assist with molecular mechanisms associated with FA formation, the Garry lab to assist with bioreactor implementation, the Zhang lab to add cardiovascular physiology expertise, and the Talkachova lab to assist with optical imaging to assess function of the 3DCTM and core facilities for mass spectrometry of ECM components and gene editing tools for modulating ILK activity. Together, this expertise will be funneled toward meeting the primary objective of the proposal via the following aims: 1) determine whether activation of integrin-linked kinase (ILK) of focal adhesions or costameres couples integrin activation to β-catenin activation via GSK3β to enable expression of genes associated with cardiomyocyte specification, 2) use 3D ECM-based model systems to identify ECM formulations supportive of endothelial differentiation and assess ILK dependence, and 3) use ECM-based 3D printing to modulate differentiation of cardiac cell types spatially in a cardiac tissue mimic. The motivation for this concept was based on extensive literature search, and results from our own experimentation; the approach was designed to insure that the interpretations of the results are subject to minimal bias and the hypotheses posed are truly tested.

项目摘要该提案的主要目的是将a）与分化结果与通过在焦点粘附（FA）触发的细胞内信号事件（FA）中参与ECM，b）3D打印 ECM和ECM相关蛋白作为成熟的细胞分布的一种手段，从而使制造厚的功能性心脏组织。该提案很重要，因为它有可能产生对患有急性和慢性损伤的人的替代组织，甚至是心脏移植。它是第一个解决焦点粘合剂明显均匀性的难题无数不同的ECM/整合素组合以及从中出现的相应各种细胞行为 ECM参与。它通过提出FA的元素，即整联蛋白连接激酶（ILK）和相关的磷酸酶充当敏感的风湿病，可以选择产生所需的行为。在这里，所需的行为是心脏差异，创新是优化的ECM公式的利用随着生物学创建3D心脏组织模拟（3DCTM），能够指导多个细胞的细胞分布具有差异化的类型。这个概念是可行的，因为Ogle实验室已长期研究了与ECM参与相关的ECM和干细胞行为，McAlpine实验室已集中从生物学到电子的一系列应用，对功能材料的3D打印以及合并这些材料。这些小组还将与坎普实验室的专家有关产生诱导的心脏祖细胞（ICPC）以填充3DCTM，即Provenzano实验室以协助通过与FA形成相关的分子机制，Garry Lab可以协助生物反应器实施，Zhang实验室以增加心血管生理学专业知识和Talkachova实验室以协助使用光学成像来评估ECM质谱的3DCTM和核心设施的功能组件和基因编辑工具用于调节ILK活动。在一起，这种专业知识将被融入通过以下目的实现提案的主要目标：1）确定是否激活焦点粘合剂或服装的整合素连接激酶（ILK）伴侣整联蛋白激活β-catenin激活通过GSK3β启用与心肌细胞规范相关的基因表达，2）使用基于3D ECM的基因识别支持内皮分化和评估的ECM公式的模型系统依赖性和3）使用基于ECM的3D打印来在空间上调节心脏细胞类型的分化心脏组织模仿。这个概念的动机是基于广泛的文献搜索和结果根据我们自己的实验；该方法旨在确保结果的解释是受到最小偏见和提出的假设的影响。